This application incorporates by reference the following U.S. patent applications: Ser. No. 09/156,318, filed Sep. 18, 1998; and Ser. No. 09/349,733, filed Jul. 8, 1999.
This application also incorporates by reference the following PCT patent applications: Ser. No. PCT/US98/23095, filed Oct. 30, 1998; Ser. No. PCT/US99/01656, filed Jan. 25, 1999; Ser. No. PCT/US99/03678, filed Feb. 19, 1999; Ser. No. PCT/US99/08410, filed Apr. 16, 1999; Ser. No. PCT/US99/16057, filed Jul. 15, 1999; and Ser. No. PCT/US99/16453, filed Jul. 21, 1999.
This application also incorporates by reference the following U.S. provisional patent applications: Ser. No. 60/094,275, filed Jul. 27, 1998; Ser. No. 60/094,276, filed Jul. 27, 1998; Ser. No. 60/094,306, filed Jul. 27, 1998; Ser. No. 60/1,00,817, filed Sep. 18, 1998; Ser. No. 60/100,951, filed Sep. 18, 1998; Ser. No. 60/104,964, filed Oct. 20, 1998; Ser. No. 60/114,209, filed Dec. 29, 1998; Ser. No. 60/116,113, filed Jan. 15, 1999; Ser. No. 60/117,278, filed Jan. 26, 1999; Ser. No. 60/119,884, filed Feb. 12, 1999; Ser. No. 60/121,229, filed Feb. 23, 1999; Ser. No. 60/124,686, filed Mar. 16, 1999; Ser. No. 60/125,346, filed Mar. 19, 1999; Ser. No. 60/126,661, filed Mar. 29, 1999; Ser. No. 60/130,149, filed Apr. 20, 1999; Ser. No. 60/132,262, filed May 3, 1999; Ser. No. 60/132,263, filed May 3, 1999; Ser. No. 60/135,284, filed May 21, 1999; Ser. No. 60/136,566, filed May 28, 1999; Ser. No. 60/138,311, filed Jun. 9, 1999; Ser. No. 60/138,438, filed Jun. 10, 1999; Ser. No. 60/138,737, filed June 11, 1999; Ser. No. 60/138,893, filed Jun. 11, 1999; and Ser. No. 60/142,721, filed Jul. 7, 1999.
This application also incorporates by reference the following publications: Max Born and Emil Wolf, Principles of Optics (6th ed. 1980); Richard P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6th ed. 1996); and Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy (1983).
The invention relates to spectroscopic assays. More particularly, the invention relates to apparatus and methods for conducting spectroscopic measurements, including absorbance, scattering, reflectance, and luminescence.
Generally speaking, spectroscopy involves the study of matter using electromagnetic radiation. Spectroscopic measurements can be separated into three broad categories: absorbance, scattering/reflectance, and emission. Absorbance assays involve relating the amount of incident light that is absorbed by a sample to the type and number of molecules in the sample. Absorbance assays are a powerful method for determining the presence and concentration of an analyte in a sample. Most commonly, absorbance is measured indirectly by studying the portion of incident light that emerges from the sample. Scattering assays are similar to absorbance in that the measurement is based on the amount of incident light which emerges or is transmitted from the sample. However, in the case of scattering, the signal increases with the number of interactions, whereas, in the case of absorbance, the signal is inversely proportional to the interactions. Emission assays look at electromagnetic emissions from a sample other than the incident light. In each case, the measurements may be broad spectrum or frequency specific depending on the particular assay.
FIG. 1 shows a schematic view of a typical absorbance experiment. Generally, absorbance measurements are made by directing incident light from a light source through a sample and through two walls of a sample container, and measuring the transmitted light using a detector. Unfortunately, this approach has a number of shortcomings. In particular, the sample container may absorb part or all of the incident and transmitted light, decreasing or eliminating the sample signal and increasing the background signal. Moreover, correcting for absorbance by the sample container requires the performance of two experiments, one involving the sample and sample container, and the other involving only the sample container.
The amount of light absorbed by a sample in an absorbance experiment generally may be described by the Beer-Lambert law:                     Absorbance        =                              -                          log              ⁡                              (                                                      I                    ⁡                                          (                      λ                      )                                                                                                  I                      0                                        ⁡                                          (                      λ                      )                                                                      )                                              =                                    ϵ              ⁡                              (                λ                )                                      ⁢            c1                                              (        1        )            
The Beer-Lambert law states that when light of wavelength xcex passes through an absorbing sample, its intensity, I, decreases exponentially. Here, I0(xcex) is the intensity of the incident light at wavelength xcex, I(xcex) is the intensity of the transmitted light, xcex5(xcex) is the decadic molar extinction coefficient, c is the concentration of absorbing molecules, and 1 is the path length. The quantity xe2x88x92log(I/I0) is termed the absorbance and is the logarithm of the reciprocal of the fraction of transmitted light.
Generally, absorbance measurements are most accurate when the absorbance is in the range 0.1-2.0, corresponding to absorption of about 20-99% of the incident light. Yet, in many biological and pharmaceutical applications, such xe2x80x9chighxe2x80x9d absorbances may be difficult to obtain, because the absorbing molecules may be expensive and/or available in small quantities. Moreover, in many biological and pharmaceutical applications, small samples are desirable, because experimental procedures may involve studying hundreds of thousands of samples, such that small samples decrease reagent costs and the overall space required.
As seen from Equation 1, absorbance may be increased by increasing the concentration of absorbing molecules. Unfortunately, this approach has a number of shortcomings. In particular, because concentration is the number of molecules per unit volume, increasing the concentration involves increasing the number of molecules and/or decreasing the volume. Yet, increasing the number of molecules is undesirable if the molecules are expensive and/or rare. Similarly, decreasing the volume is undesirable because it may decrease the path length and so decrease absorbance.
Also as seen from Equation 1, absorbance may be increased by increasing the path length. Unfortunately, this approach also has a number of shortcomings. In particular, increasing the path length may involve increasing the volume of sample, and hence increasing the number of molecules and the overall space required. Alternatively, increasing the path length may involve decreasing the cross section of the sample, decreasing signal.
Scattering assays can be used to detect the motion, size, concentration, aggregation state, and other properties of molecules in a sample. For example, by looking at the spectral spread of scattered light, it is possible to determine the average velocity of scattering particles in a sample. By observing the intensity of scattered light, the concentration of scattering objects can be measured. By observing the angular distribution of scattered light, various physical characteristics of scattering molecules can be deduced.
Luminescence is the emission of light from excited electronic states of atoms or molecules. Luminescence generally refers to all kinds of light emission, except incandescence, and may include photoluminescence, chemiluminescence, and electrochemiluminescence, among others. In photoluminescence, including fluorescence and phosphorescence, the excited electronic state is created by the absorption of electromagnetic radiation. In chemiluminescence, which includes bioluminescence, the excited electronic state is created by a transfer of chemical energy. In electrochemiluminescence, the excited electronic state is created by an electrochemical process.
Luminescence assays are assays that use luminescence emissions from luminescent analytes to study the properties and environment of the analyte, as well as binding reactions and enzymatic activities involving the analyte, among others. In this sense, the analyte may act as a reporter to provide information about another material or target substance that may be the focus of the assay. Luminescence assays may use various aspects of the luminescence, including its intensity, polarization, and lifetime, among others. Luminescence assays also may use time-independent (steady-state) and/or time-dependent (time-resolved) properties of the luminescence. Steady-state assays generally are less complicated than time-resolved assays, but generally yield less information.
A. Intensity Assays
Luminescence intensity assays involve monitoring the intensity (or amount) of light emitted from a composition. The intensity of emitted light will depend on the extinction coefficient, quantum yield, and number of the luminescent analytes in the composition, among others. These quantities, in turn, will depend on the environment of the analyte, among others, including the proximity and efficacy of quenchers and energy transfer partners. Thus, luminescence intensity assays may be used to study binding reactions, among other applications.
B. Polarization Assays
Luminescence polarization assays involve the absorption and emission of polarized light, and typically are used to study molecular rotation. (Polarization describes the direction of light""s electric field, which generally is perpendicular to the direction of light""s propagation.)
FIG. 2 is a schematic view showing how luminescence polarization is affected by molecular rotation. In a luminescence polarization assay, specific molecules 30 within a composition 32 are labeled with one or more luminophores. The composition then is illuminated with polarized excitation light, which preferentially excites luminophores having absorption dipoles aligned parallel to the polarization of the excitation light. These molecules subsequently decay by preferentially emitting light polarized parallel to their emission dipoles. The extent to which the total emitted light is polarized depends on the extent of molecular reorientation during the time interval between luminescence excitation and emission, which is termed the luminescence lifetime, xcfx84. The extent of molecular reorientation in turn depends on the luminescence lifetime and the size, shape, and environment of the reorienting molecule. Thus, luminescence polarization assays may be used to quantify binding reactions and enzymatic activity, among other applications. In particular, molecules rotate via diffusion with a rotational correlation time xcfx84rot that is proportional to their size. Thus, during their luminescence lifetime, relatively large molecules will not reorient significantly, so that their total luminescence will be relatively polarized. In contrast, during the same time interval, relatively small molecules will reorient significantly, so that their total luminescence will be relatively unpolarized.
The relationship between polarization and intensity is expressed by the following equation:                     P        =                                            I              ∥                        -                          I              ⊥                                                          I              ∥                        +                          I              ⊥                                                          (        2        )            
Here, P is the polarization, I∥ is the intensity of luminescence polarized parallel to the polarization of the excitation light, and Ixe2x8axa5 is the intensity of luminescence polarized perpendicular to the polarization of the excitation light. P generally varies from zero to one-half for randomly oriented molecules (and zero to one for aligned molecules). If there is little rotation between excitation and emission, I∥ will be relatively large, Ixe2x8axa5 will be relatively small, and P will be close to one-half (P may be less than one-half even if there is no rotation; for example, P will be less than one if the absorption and emission dipoles are not parallel.) In contrast, if there is significant rotation between absorption and emission, Ii| will be comparable to Ixe2x8axa5, and P will be close to zero. Polarization often is reported in milli-P (mP) units (1000xc3x97P), which for randomly oriented molecules will range between 0 and 500, because P will range between zero and one-half.
Polarization also may be described using other equivalent quantities, such as anisotropy. The relationship between anisotropy and intensity is expressed by the following equation:                     r        =                                            I              ∥                        -                          I              ⊥                                                          I              ∥                        +                          2              ⁢                              I                ⊥                                                                        (        3        )            
Here, r is the anisotropy. Polarization and anisotropy include the same information, although anisotropy may be more simply expressed for systems containing more than one luminophore. In the description and claims that follow, these terms may be used interchangeably, and a generic reference to one should be understood to imply a generic reference to the other.
The relationship between polarization and rotation is expressed by the Perrin equation:                               (                                    1              P                        -                          1              3                                )                =                              (                                          1                                  P                  0                                            -                              1                3                                      )                    ·                      (                          1              +                              τ                                  τ                  rot                                                      )                                              (        4        )            
Here, P0 is the polarization in the absence of molecular motion (intrinsic polarization), xcfx84 is the luminescence lifetime (inverse decay rate) as described above, and xcfx84rot is the rotational correlation time (inverse rotational rate) as described above.
The Perrin equation shows that luminescence polarization assays are most sensitive when the luminescence lifetime and the rotational correlation time are similar. Rotational correlation time is proportional to molecular weight, increasing by about 1 nanosecond for each 2,400 Dalton increase in molecular weight (for a spherical molecule). For shorter lifetime luminophores, such as fluorescein, which has a luminescence lifetime of roughly 4 nanoseconds, luminescence polarization assays are most sensitive for molecular weights less than about 40,000 Daltons. For longer lifetime probes, such as Ru(bpy)2dcbpy (ruthenium 2,2xe2x80x2-dibipyridyl 4,4xe2x80x2-dicarboxyl-2,2xe2x80x2-bipyridine), which has a lifetime of roughly 400 nanoseconds, luminescence polarization assays are most sensitive for molecular weights between about 70,000 Daltons and 4,000,000 Daltons.
C. Time-Resolved Assays
Time-resolved assays involve measuring the time course of luminescence emission. Time-resolved assays may be conducted in the time domain or in the frequency domain, both of which are functionally equivalent. In a time-domain measurement, the time course of luminescence is monitored directly. Typically, a composition containing a luminescent analyte is illuminated using a narrow pulse of light, and the time dependence of the intensity of the resulting luminescence emission is observed, although other protocols also may be used. For a simple molecule, the luminescence commonly follows a single-exponential decay.
In a frequency-domain measurement, the time course of luminescence is monitored indirectly, in frequency space. Typically, the composition is illuminated using light whose intensity is modulated sinusoidally at a single modulation frequency f, although other protocols (such as transforming time-domain data into the frequency domain) also may be used. The intensity of the resulting luminescence emission is modulated at the same frequency as the excitation light. However, the emission will lag the excitation by a phase angle (phase) xcfx86, and the intensity of the emission will be demodulated relative to the intensity of the excitation by a demodulation factor (modulation) M.
FIG. 3 shows the relationship between emission and excitation in a single-frequency frequency-domain experiment. The phase xcfx86 is the phase difference between the excitation and emission. The modulation M is the ratio of the AC amplitude to the DC amplitude for the emission, relative to the ratio of the AC amplitude to the DC amplitude for the excitation. The phase and modulation are related to the luminescence lifetime xcfx84 by the following equations:
xcfx89xcfx84=tan(xcfx86)xe2x80x83xe2x80x83(5)
                    ωτ        =                                            1                              M                2                                      -            1                                              (        6        )            
Here xcfx89 is the angular modulation frequency, which equals 2xcfx80 times the modulation frequency. For maximum sensitivity, the angular modulation frequency should be roughly the inverse of the luminescence lifetime. Lifetimes of interest in high-throughput screening vary from less than 1 nanosecond to greater than 1 milliseconds. Therefore, instruments for high-throughput screening should be able to cover modulation frequencies from less than about 200 Hz to about 200 MHz.
D. Strengths and Weaknesses of Luminescence Assays
Luminescence methods have several significant potential strengths. First, luminescence methods may be very sensitive, because modern detectors, such as photomultiplier tubes (PMTs) and charge-coupled devices (CCDs), can detect very low levels of light. Second, luminescence methods may be very selective, because the luminescence signal may come almost exclusively from the luminophore.
Luminescence assays also have several significant potential weaknesses. First, luminescence from the analyte might be perturbed in some way, distorting results. For example, if a luminescent analyte binds to the walls of a sample holder during a luminescence polarization assay, the analyte will be unable to rotate, spuriously increasing the polarization. Second, luminescence may arise from sources other than the analyte, contaminating the signal. For example, luminescence may arise from the sample holder, including glass coverslips and plastic microplates.